The NADPH oxidase proteins, or Nox, are tissue-specific modular proteins of very wide occurrence in eukaryotes. They share a catalytic core consisting of an FNR (flavin-containing ferredoxin NADPH oxidoreductase-like) domain and an FRD (ferric reductase domain), bound to the membrane, fixing 2 haems (Nox 1 to 4). They may also have, as Nox5, a calmodulin domain and in addition, as in the case of Duox1 and Duox2, an additional transmembrane segment and a domain of the peroxidase type at the N-terminal end (Lambeth J D, NOX enzymes and the biology of reactive oxygen, Nature Reviews, 2004, Immunology, 4:181-189). Although the two domains FNR and cytochrome b are found in prokaryotes, pure and simple sequence alignments have never allowed Nox or Fre proteins to be detected in the bacterial genomes.
In terms of function, in addition to Nox2, which is found both in phagocytes, in which it is involved in immune defence, and in the cardiac tissues, in which it plays a role in muscle contraction, the Nox enzymes are involved in multiple processes such as cellular proliferation, apoptosis, or intra- and intercellular communication. Moreover, the Duox enzymes are involved in the synthesis of the thyroid hormones (Prosser B L et al., X-ROS signaling: rapid mechano-chemo transduction in heart, 2011, Science, 333:1440-1445; De Deken X et al., Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family, The Journal of Biological Chemistry, 275:23227-23233). In yeast, the ferric reductases (Fre) for their part are involved in the reduction of ferric ions to ferrous ions during iron import.
In a controlled manner, the Nox proteins lead to the production of reactive oxygen species (ROS), such as the superoxide ions O2.−, hydrogen peroxide (H2O2), or the OH. hydroxyl radical. Recent accumulation of data indicates that the ROS have a much more subtle role than was initially attributed in the immune system. Their importance in molecular and cellular mechanisms such as signalling, chemical reactivity, or toxicity induced by the redox potential of the species produced, therefore places the Nox proteins henceforth in the rank of high-added-value therapeutic targets.
To identify new medicaments capable of specifically targeting this protein family, the pharmaceutical industry screens small molecules to determine their effects with respect to Nox proteins. These small molecules are then improved by organic synthesis in order to optimize their effects.
Now, taking into account the eukaryotic origin of the Nox proteins, their capacity as membrane proteins, their modes of regulation, and their specific post-translational modifications (Nox1-4, Nox5 and Duox1-2), current pharmaceutical research must tackle the difficulties inherent in the very nature of these Nox proteins. In other words, it is difficult to produce in sufficient quantities a highly regulated glycosylated membrane system, with multiple components.
For carrying out screening assays, it is however necessary on the one hand to obtain functional Nox proteins to evaluate the actual impact of the molecules tested, and on the other hand to produce sufficient quantities of Nox proteins so as to be able to test a large number of molecules. Now, the Nox proteins produced in large quantities are mainly inactive, whereas the functional Nox proteins themselves are obtained in insignificant quantities. This has the effect of making their production cost too high to envisage screening assays on a large scale.
Currently, human Nox proteins have already been produced in cells of the immune system (Taylor R M et al., Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry, J Biol Chem 2006, Dec. 1, 281(48): 37045-56; Lord C I et al., Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b, J Immunol Methods, 2008, Jan. 1, 329(1-2):201-7) and various attempts at heterologous expression have also been envisaged, but without success (de Mendez I, Leto T L, Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system, Blood, 1995, Feb. 15, 85(4):1104-10; Price M O et al. Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system. Blood, 2002, Apr. 15, 99(8):2653-61; Zhen L et al., Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci USA, 1993 Nov. 1, 90(21):9832-6).
Mammalian Nox proteins have in particular been expressed heterologously in various eukaryotic organisms, such as in insect cells (Rotrosen D et al., Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins, J Biol Chem, 1993, Jul. 5, 268(19):14256-60) or in yeast (Ostuni M A et al., Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system. Biochim Biophys Acta, 2010 June, 1798(6):1179-88).
However, none of these approaches has made it possible to produce functional Nox proteins in sufficient quantities to envisage high-throughput screening.
Moreover, heterologous expression systems have also been tested in attempts to produce eukaryotic Nox proteins in prokaryotic organisms, in particular in the bacterium Escherichia coli, but only truncated forms of the eukaryotic Nox proteins have been produced (Han C H et al., Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity, J Biochem, 2001 April 129(4):513-20; Nisimoto Y et al. Activation of the flavoprotein domain of gp91 phox upon interaction with N-terminal p67phox (1-210) and the Rac complex, Biochemistry, 2004, Jul. 27, 43(29):9567-75).